This work shows the possibility of combining the high sensitivity of genetically-modified Drosophila melanogaster acetylcholinesterase (B394) with the ability of phosphotriesterase (PTE) to hydrolyse organophosphate compounds, in the aim of developing a biosensor selective to two insecticides of interest: chlorpyrifos and chlorfenvinfos. The studies clearly demonstrate that chlorfenvinfos is a substrate that acts as competitive inhibitor of PTE, therefore preventing the efficient hydrolysis of other pesticides, including chlorpyrifos. A bi-enzymatic sensor was designed by immobilizing both B394 and PTE in a polyvinylalcohol matrix. The sensor was shown to be able to discriminate between chlorpyrifos and chlorfenvinfos inhibitions.